Enantioselective de novo synthesis of 14-hydroxy-6-oxomorphinans.

The enantioselective de novo synthesis of pharmacologically important 14-hydroxy-6-oxomorphinans is described. 4,5-Desoxynaltrexone and 4,5-desoxynaloxone were prepared using this route and their biological activities against the opioid receptors were measured.

Searching for Synthetic Opioid Rescue Agents: Identification of a Potent Opioid Agonist with Reduced Respiratory Depression.

While in the process of designing more effective synthetic opioid rescue agents, we serendipitously identified a new chemotype of potent synthetic opioid. Here, we report that conformational constraint of a piperazine ring converts a mu opioid receptor (MOR) antagonist into a potent MOR agonist. The prototype of the series, which we have termed atoxifent (2), possesses potent in vitro agonist activity. In mice, atoxifent displayed long-lasting antinociception that was reversible with naltrexone. Repeated dosing of atoxifent produced antinociceptive tolerance and a level of withdrawal like that of fentanyl. In rats, while atoxifent produced complete loss of locomotor activity like fentanyl, it failed to produce deep respiratory depression associated with fentanyl-induced lethality. Assessment of brain biodistribution demonstrated ample distribution of atoxifent into the brain with a Tmax of approximately 0.25 h. These results indicate enhanced safety for atoxifent-like molecules compared to fentanyl.

Design, synthesis, and preliminary evaluation of a potential synthetic opioid rescue agent.

BACKGROUND: One of the most prominent opioid analgesics in the United States is the high potency agonist fentanyl. It is used in the treatment of acute and chronic pain and as an anesthetic adjuvant. When used inappropriately, however, ingestion of just a few milligrams of fentanyl or other synthetic opioid can cause opioid-induced respiratory depression (OIRD), often leading to death. Currently, the treatment of choice for OIRD is the opioid receptor antagonist naloxone. Recent reports, however, suggest that higher doses or repeated dosing of naloxone (due to recurrence of respiratory depression) may be required to reverse fully fentanyl-induced respiratory depression, rendering this treatment inadequate. To combat this synthetic opioid overdose crisis, this research aims at identifying a novel opioid reversal agent with enhanced efficacy towards fentanyl and other synthetic opioids. METHODS: A series of naltrexone analogues were characterized for their ability to antagonize the effects of fentanyl in vitro utilizing a modified forskolin-induced cAMP accumulation assay. Lead analogue 29 was chosen to undergo further PK studies, followed by in vivo pharmacological analysis to determine its ability to antagonize opioid-induced antinociception in the hot plate assay. RESULTS: A series of potent MOR antagonists were identified, including the highly potent analogue 29 (IC50 = 2.06 nM). Follow-up PK studies revealed 29 to possess near 100% bioavailability following IP administration. Brain concentrations of 29 surpassed plasma concentrations, with an apparent terminal half-life of ~ 80 min in mice. In the hot plate assay, 29 dose-dependently (0.01-0.1 mg/kg; IP) and fully antagonized the antinociception induced by oxycodone (5.6 mg/kg; IP). Furthermore, the dose of 29 that is fully effective in preventing oxycodone-induced antinociception (0.1 mg/kg) was ineffective against locomotor deficits caused by the KOR agonist U50,488. CONCLUSIONS: Methods have been developed that have utility to identify enhanced rescue agents for the treatment of OIRD. Analogue 29, possessing potent MOR antagonist activity in vitro and in vivo, provides a promising lead in our search for an enhanced synthetic opioid rescue agent.

Exploring naltrexamine derivatives featuring azaindole moiety via nitrogen-walk approach to investigate their in vitro pharmacological profiles at the mu opioid receptor.

In the present work, we reported the application of a nitrogen-walk approach on developing a series of novel opioid ligands containing an azaindole moiety at the C6-position of the epoxymorphinan skeleton. In vitro study results showed that introducing a nitrogen atom around the indole moiety not only retained excellent binding affinity, but also led to significant functional switch at the mu opioid receptor (MOR). Further computational investigations provided corroborative evidence and plausible explanations of the results of the in vitro studies. Overall, our current work implemented a series of novel MOR ligands with high binding affinity and considerably low efficacy, which may shed light on rational design of low efficacy MOR ligands for opioid use disorder therapeutics.

Synthesis and Characterization of Azido Aryl Analogs of IBNtxA for Radio-Photoaffinity Labeling Opioid Receptors in Cell Lines and in Mouse Brain.

Mu opioid receptors (MOR-1) mediate the biological actions of clinically used opioids such as morphine, oxycodone, and fentanyl. The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, generating multiple splice variants. One type of splice variants are truncated variants containing only six transmembrane domains (6TM) that mediate the analgesic action of novel opioid drugs such as 3'-iodobenzoylnaltrexamide (IBNtxA). Previously, we have shown that IBNtxA is a potent analgesic effective in a spectrum of pain models but lacks many side-effects associated with traditional opiates. In order to investigate the targets labeled by IBNtxA, we synthesized two arylazido analogs of IBNtxA that allow photolabeling of mouse mu opioid receptors (mMOR-1) in transfected cell lines and mMOR-1 protein complexes that may comprise the 6TM sites in mouse brain. We demonstrate that both allyl and alkyne arylazido derivatives of IBNtxA efficiently radio-photolabeled mMOR-1 in cell lines and MOR-1 protein complexes expressed either exogenously or endogenously, as well as found in mouse brain. In future, design and application of such radio-photolabeling ligands with a conjugated handle will provide useful tools for further isolating or purifying MOR-1 to investigate site specific ligand-protein contacts and its signaling complexes.

Effects of N-Substituents on the Functional Activities of Naltrindole Derivatives for the δ Opioid Receptor: Synthesis and Evaluation of Sulfonamide Derivatives.

We have recently reported that N-alkyl and N-acyl naltrindole (NTI) derivatives showed activities for the δ opioid receptor (DOR) ranging widely from full inverse agonists to full agonists. We newly designed sulfonamide-type NTI derivatives in order to investigate the effects of the N-substituent on the functional activities because the side chain and S=O part in the sulfonamide moiety located in spatially different positions compared with those in the alkylamine and amide moieties. Among the tested compounds, cyclopropylsulfonamide 9f (SYK-839) was the most potent full inverse agonist for the DOR, whereas phenethylsulfonamide 9e (SYK-901) showed full DOR agonist activity with moderate potency. These NTI derivatives are expected to be useful compounds for investigation of the molecular mechanism inducing these functional activities.

Bioresponsive nanostructured systems for sustained naltrexone release and treatment of alcohol use disorder: Development and biological evaluation.

In this study, microemulsions capable of transforming into nanostructured hexagonal phase gels in vivo upon uptake of biological fluids for naltrexone prolonged release were investigated as a strategy for management of alcohol use disorder (AUD). Microemulsions were prepared using monoolein, tricaprylin, water and propylene glycol; after preliminary characterization, one formulation was selected, which contained 55% of monoolein-tricaprylin (M-55). This microemulsion displayed size below 200 nm and Newtonian rheological behavior. Liquid crystalline gels formed in vitro upon 8 h of contact with water following a second order kinetics. After 120 h, <50% of naltrexone was released in vitro independently on drug loading (5 or 10%). In vivo, gels formed within 24 h of M-55 subcutaneous administration, and persisted locally for over 30 days providing slow release of the fluorescent marker Alexa fluor compared to a solution. Using the conditioned place preference paradigm, a test used to measure drug's rewarding effects, a single dose of M-55 containing 5% naltrexone reduced the time spent in the ethanol-paired compartment by 1.8-fold compared to saline; this effect was similar to that obtained with daily naltrexone injections, demonstrating the formulation efficacy and its ability to reduce dosing frequency. A more robust effect was observed following administration of M-55 containing 10% of naltrexone, which was compatible with aversion. These results support M-55 as a platform for sustained release of drugs that can be further explored for management of AUD to reduce dosing frequency and aid treatment adherence.

Investigation of Inactive-State κ Opioid Receptor Homodimerization via Single-Molecule Microscopy Using New Antagonistic Fluorescent Probes.

Opioid receptors (ORs) are among the best-studied G protein-coupled receptors due to their involvement in neurological disorders and important role in pain treatment. Contrary to the classical monomeric model, indirect evidence suggests that ORs might form dimers, which could be endowed with a distinct pharmacological profile, and, thus, be targeted to develop innovative pharmacological therapies. However, direct evidence for the spontaneous formation of OR dimers in living cells under physiological conditions is missing. Despite a growing interest in the κ opioid receptor (KOR), KOR-selective fluorescent probes are particularly scarce in the literature. Herein, we present the first set of fluorescent KOR-selective probes with antagonistic properties. Two of these were employed in single-molecule microscopy (SMM) experiments to investigate KOR homodimerization, localization, and trafficking. Our findings indicate that most KORs labeled with the new fluorescent probes are present as apparently freely diffusing monomers on the surface of a simple cell model.

Antitrichomonal activity of δ opioid receptor antagonists, 7-benzylidenenaltrexone derivatives.

The 7-benzylidenenaltrexone (BNTX) derivatives 2a-v, 3a-c, 13a-c, and 14a were synthesized from naltrexone (1) and evaluated for their antitrichomonal activity. The structure-activity-relationship studies found that 4-iodo-BNTX (2g) showed the highest activity (IC50=10.5µM) and the affinity for the opioid receptor was less important for antitrichomonal activity against Trichomonas vaginalis. The morphinan skeleton bearing both the double bond for a Michael acceptor and the phenolic hydroxy group would be a specific template for development of antitrichomonal agents. In addition, the mechanism of the antitrichomonal activity of the BNTX derivatives may differ from that of the standard drug, metronidazole.

New opioid receptor antagonist: Naltrexone-14-O-sulfate synthesis and pharmacology.

Opioid antagonists, naloxone and naltrexone have long been used in clinical practice and research. In addition to their low selectivity, they easily pass through the blood-brain barrier. Quaternization of the amine group in these molecules, (e.g. methylnaltrexone) results in negligible CNS penetration. In addition, zwitterionic compounds have been reported to have limited CNS access. The current study, for the first time gives report on the synthesis and the in vitro [competition binding, G-protein activation, isolated mouse vas deferens (MVD) and mouse colon assay] pharmacology of the zwitterionic compound, naltrexone-14-O-sulfate. Naltrexone, naloxone, and its 14-O-sulfate analogue were used as reference compounds. In competition binding assays, naltrexone-14-O-sulfate showed lower affinity for µ, δ or κ opioid receptor than the parent molecule, naltrexone. However, the µ/κ opioid receptor selectivity ratio significantly improved, indicating better selectivity. Similar tendency was observed for naloxone-14-O-sulfate when compared to naloxone. Naltrexone-14-O-sulfate failed to activate [35S]GTPγS-binding but inhibit the activation evoked by opioid agonists (DAMGO, Ile5,6deltorphin II and U69593), similarly to the reference compounds. Schild plot constructed in MVD revealed that naltrexone-14-O-sulfate acts as a competitive antagonist. In mouse colon, naltrexone-14-O-sulfate antagonized the inhibitory effect of morphine with lower affinity compared to naltrexone and higher affinity when compared to naloxone or naloxone-14-O-sulfate. In vivo (mouse tail-flick test), subcutaneously injected naltrexone-14-O-sulfate antagonized morphine's antinociception in a dose-dependent manner, indicating it's CNS penetration, which was unexpected from such zwitter ionic structure. Future studies are needed to evaluate it's pharmacokinetic profile.

Exploration of bivalent ligands targeting putative mu opioid receptor and chemokine receptor CCR5 dimerization.

Modern antiretroviral therapies have provided HIV-1 infected patients longer lifespans and better quality of life. However, several neurological complications are now being seen in these patients due to HIV-1 associated injury of neurons by infected microglia and astrocytes. In addition, these effects can be further exacerbated with opiate use and abuse. One possible mechanism for such potentiation effects of opiates is the interaction of the mu opioid receptor (MOR) with the chemokine receptor CCR5 (CCR5), a known HIV-1 co-receptor, to form MOR-CCR5 heterodimer. In an attempt to understand this putative interaction and its relevance to neuroAIDS, we designed and synthesized a series of bivalent ligands targeting the putative CCR5-MOR heterodimer. To understand how these bivalent ligands may interact with the heterodimer, biological studies including calcium mobilization inhibition, binding affinity, HIV-1 invasion, and cell fusion assays were applied. In particular, HIV-1 infection assays using human peripheral blood mononuclear cells, macrophages, and astrocytes revealed a notable synergy in activity for one particular bivalent ligand. Further, a molecular model of the putative CCR5-MOR heterodimer was constructed, docked with the bivalent ligand, and molecular dynamics simulations of the complex was performed in a membrane-water system to help understand the biological observation.

6ß-N-Heterocyclic Substituted Naltrexamine Derivative BNAP: A Peripherally Selective Mixed MOR/KOR Ligand.

The 6ß-N-heterocyclic naltrexamine derivative, NAP, has been demonstrated to be a peripherally selective mu opioid receptor modulator. To further improve peripheral selectivity of this highly potent ligand, its pyridal ring was quaterinized with benzyl bromide to produce BNAP. In radioligand binding assay, the Ki of BNAP for MOR was 0.76 ± 0.09 nM and was >900-fold more selective for MOR than DOR. The Ki for KOR was 3.46 ± 0.05 nM. In [(35)S]GTPγS ligand stimulated assay, BNAP showed low agonist efficacy with 14.6% of the maximum response of DAMGO with an EC50 of 4.84 ± 0.6 nM. However, unlike its parent compound NAP, BNAP displayed partial agonist activity at KOR with % maximum response at 45.9 ± 1.7% of U50,488H. BNAP did not reverse morphine-induced antinociception when administered subcutaneously but did antagonize when administered intracerebroventricularly. BNAP antagonized morphine-induced contractions of the circular muscle in mice colon. BNAP inhibition of field-stimulated contractions in longitudinal muscle strips for the guinea-pig ileum were also blocked by nor-BNI, a kappa opioid receptor antagonist. BNAP induced inhibition of acetic acid induced abdominal stretching in chronic morphine treated mice. These findings suggest that BNAP is a dual MOR antagonist/KOR agonist and may have functional use in irritable bowel patients.

Synthesis and evaluation of novel opioid ligands with a C-homomorphinan skeleton.

As the reports about C-homomorphinans with the seven-membered C-ring are much fewer than those of morphinan derivatives with a six-membered C-ring, we attempted to synthesize C-homomorphinan derivatives and to evaluate their opioid activities. C-Homomorphinan 5 showed sufficient binding affinities to the opioid receptors. C-Homomorphinan derivatives possessing the δ address moiety such as indole (NTI-type), quinoline, or benzylidene (BNTX-type) functionalities showed the strongest binding affinities for the δ receptor among the three types of opioid receptors, which indicated that the C-homomorphinan skeleton sufficiently functions as a message-part in the ligand. Although NTI-type compound 8 and quinoline compound 9 with C-homomorphinan scaffold exhibited lower affinities and selectivities for the δ receptor than the corresponding morphinan derivatives did, both the binding affinity and selectivity for the δ receptor of BNTX-type compound 12 with a seven-membered C-ring were improved compared with the corresponding compounds with a six-membered C-ring including BNTX itself. BNTX-Type compound 12 was the most selective δ receptor antagonist among the tested compounds.

Investigation of 7-benzylidenenaltrexone derivatives as a novel structural antitrichomonal lead compound.

We evaluated antitrichomonal effects of δ opioid receptor (DOR) agonists and antagonists. Although all the agonists were inactive, the DOR antagonists BNTX (2a) and its derivatives 2b-d showed antitrichomonal activity with MIC of 20-40 µM. In addition, the development of a more effective synthetic method for the BNTX derivatives was achieved by using the Knoevenagel condensation.

Synthesis and in vitro stability of amino acid prodrugs of 6-ß-naltrexol for microneedle-enhanced transdermal delivery.

A small library of amino acid ester prodrugs of 6-ß-naltrexol (NTXOL, 1) was prepared in order to investigate the candidacy of these prodrugs for microneedle-enhanced transdermal delivery. Six amino acid ester prodrugs were synthesized (6a-f). 6b, 6d, and 6 e were stable enough at skin pH (pH 5.0) to move forward to studies in 50% human plasma. The lead compound (6 e) exhibited the most rapid bioconversion to NTXOL in human plasma (t1/2 = 2.2 ± 0.1h).

Synthesis of enantiopure 10-nornaltrexones in the search for Toll-like receptor 4 antagonists and opioid ligands.

10-Nornaltrexones (3-(cyclopropylmethyl)-4a,9-dihydroxy-2,3,4,4a,5,6-hexahydro-1H-benzofuro[3,2-e]isoquinolin-7(7aH)-one, 1) have been underexploited in the search for better opioid ligands, and their enantiomers have been unexplored. The synthesis of trans-isoquinolinone 2 (4-aH, 9-O-trans-9-methoxy-3-methyl-2,3,4,4a,5,6-hexahydro-1H-benzofuro[3,2-e]isoquinolin-7(7aH)-one) was achieved through a nonchromatographic optimized synthesis of the intermediate pyridinyl compound 12. Optical resolution was carried out on 2, and each of the enantiomers were used in efficient syntheses of the "unnatural" 4aR,7aS,12bR-(+)-1) and its "natural" enantiomer (-)-1. Addition of a 14-hydroxy (the 4a-hydroxy) group in the enantiomeric isoquinolinones, (+)- and (-)-2), gave (+)- and (-)-10-nornaltrexones. A structurally unique tetracyclic enamine, (12bR)-7,9-dimethoxy-3-methyl-1,2,3,7-tetrahydro-7,12b-methanobenzo[2,3]oxocino[5,4-c]pyridine, was found as a byproduct in the syntheses and offers a different opioid-like skeleton for future study.

Synthesis of [15, 16- ³H] beta-funaltrexamine.

Beta-funaltrexamine is a unique irreversible antagonist for the mu-opiate receptor and would be useful as a tritiated radioligand. Starting from high specific activity [15, 16-³H] naltrexone, [15, 16-³H] beta-funaltrexamine was synthesized and characterized by means of a two-step reductive amination-acylation process.

Design, synthesis, and biological evaluation of 14-heteroaromatic-substituted naltrexone derivatives: pharmacological profile switch from mu opioid receptor selectivity to mu/kappa opioid receptor dual selectivity.

On the basis of a mu opioid receptor (MOR) homology model and the isosterism concept, three generations of 14-heteroaromatically substituted naltrexone derivatives were designed, synthesized, and evaluated as potential MOR-selective ligands. The first-generation ligands appeared to be MOR-selective, whereas the second and the third generation ones showed MOR/kappa opioid receptor (KOR) dual selectivity. Docking of ligands 2 (MOR selective) and 10 (MOR/KOR dual selective) to the three opioid receptor crystal structures revealed a nonconserved-residue-facilitated hydrogen-bonding network that could be responsible for their distinctive selectivity profiles. The MOR/KOR dual-selective ligand 10 showed no agonism and acted as a potent antagonist in the tail-flick assay. It also produced less severe opioid withdrawal symptoms than naloxone in morphine-dependent mice. In conclusion, ligand 10 may serve as a novel lead compound to develop MOR/KOR dual-selective ligands, which might possess unique therapeutic value for opioid addiction treatment.

Binding mode characterization of 6α- and 6ß-N-heterocyclic substituted naltrexamine derivatives via docking in opioid receptor crystal structures and site-directed mutagenesis studies: application of the 'message-address' concept in development of mu opioid receptor selective antagonists.

Highly selective opioid receptor antagonists are essential pharmacological probes in opioid receptor structural characterization and opioid agonist functional studies. Currently, there is no highly selective, nonpeptidyl and reversible mu opioid receptor antagonist available. Among a series of naltrexamine derivatives that have been designed and synthesized, two compounds, NAP and NAQ, were previously identified as novel leads for this purpose based on their in vitro and in vivo pharmacological profiles. Both compounds displayed high binding affinity and selectivity to the mu opioid receptor. To further study the interaction of these two ligands with the three opioid receptors, the recently released opioid receptor crystal structures were employed in docking studies to further test our original hypothesis that the ligands recognize a unique 'address' domain in the mu opioid receptor involving Trp318 that facilitates their selectivity. These modeling results were supported by site-directed mutagenesis studies on the mu opioid receptor, where the mutants Y210A and W318A confirmed the role of the latter in binding. Such work not only enriched the 'message-address' concept, also facilitated our next generation ligand design and development.